VDAC
Voltage-dependent anion channel 1 (VDAC1; mitochondrial porin) is a pore-forming protein found in the outer mitochondrial membrane (OMM) in all eukaryotic cells controlling the fluxes of ions and metabolites between the mitochondria and the cytosol. VDAC is recognized as a key protein in mitochondria-mediated apoptosis due to its function in the release of apoptotic proteins located in the inter-membranal space and its interaction with apoptotic proteins (Shoshan-Barmatz et al, 2006). VDAC also serves as binding sites for several cytosolic enzymes and mitochondrial intermembranal space proteins, including hexokinase, creatine kinase and glycerol kinase.
Three mammalian isoforms of VDAC are known, VDAC1, VDAC2, VDAC3, where VDAC1 is the major isoform expressed in mammalian cells. Blachly-Dysion et al (1993) disclosed the cloning and functional expression in yeast of two human VDAC isoforms, VDAC1 and VDAC2. U.S. Pat. No. 5,780,235 discloses HACH (human voltage-dependent anion channel), subsequently identified as VDAC3. That patent provides genetically engineered expression vectors, host cells containing the vector, a method for producing HACH and a method for identifying pharmaceutical compositions inhibiting the expression and activity of HACH and for the use of such compositions for the treatment of cancer and proliferative diseases.
Mitochondria play an important role in the regulation of apoptotic cell death. The release of apoptogenic intermediates such as cytochrome c from the intermembranal space into the cytoplasm of a cell initiates a cascade of caspase activation that executes the cell death program. Substantial evidence links VDAC to apoptosis and suggests that VDAC is a critical player in the release of apoptogenic proteins from mitochondria in mammalian cells (Shoshan-Barmatz and Gincel, 2003; Shoshan-Barmatz et al, 2006).
It is well known that effective exchange of metabolites between mitochondria and the cytoplasm is essential for cell physiology. The key step of the exchange is transport across the outer mitochondrial membrane (OMM), which is mediated by VDAC (Colombini, 2004). The permeability of VDAC is regulated to adjust its activity to the actual cell's needs (Shoshan-Barmatz et al, 2006).
Certain compositions related to VDAC and use thereof for either inhibiting or inducing apoptosis are known in the art. International Patent Application No. PCT/IL2006/000311, to some of the inventors of the present application discloses VDAC1 specific apoptosis inducing peptides, which are useful in the treatment of diseases associated with aberrant apoptosis.
US Patent Application Publication No. 20050234116 discloses small molecule compounds with utility as VDAC regulators, in particular as apoptosis suppressors.
Gene Silencing
The down regulation of specific gene expression in a cell can be effected by oligonucleic acids using techniques known as antisense therapy and RNA interference (RNAi).
Antisense therapy refers to the process of inactivating target DNA or mRNA sequences through the use of complementary DNA or RNA oligonucleotides, thereby inhibiting gene transcription or translation. An antisense molecule can be single stranded, double stranded or triple helix.
RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in eukaryotic cells mediated by RNA fragments. The process of post-transcriptional gene silencing is thought to be an evolutionarily conserved defense mechanism used by cells to prevent the expression of foreign genes and is commonly shared by diverse organisms.
RNAi can be induced in a cell by different species of double stranded RNA molecules, including short interfering RNA (siRNA) and short hairpin RNA (shRNA). In RNAi, one strand of double-stranded RNA molecule has the ribo-oligonucleotide sequence that is identical or substantially identical to the nucleotide sequence in the targeted mRNA transcript; the second strand of RNA has a complementary sequence to that in the target mRNA. Without wishing to be bound to theory, it is accepted that once the siRNAs are introduced into a cell or are generated from longer dsRNAs in the cell by the RNaseIII like enzyme, the siRNA associates with a protein complex, known as the RNA-induced silencing complex (RISC). The RISC then guides the small double stranded siRNA to the mRNA where the two strands of the double stranded siRNA separate, the antisense strand associates with the mRNA and a nuclease cleaves the mRNA at the site where the antisense strand of the siRNA binds (Hammond et al., 2005). The mRNA is subsequently further degraded by cellular nucleases. siRNAs appear to suppress gene expression without producing non-specific cytotoxic responses. Short hairpin RNAs have been shown to be potent RNAi triggers and in some instances maybe more effective than siRNA molecules (Siolas, et al., 2005). shRNAs may be produced by chemical synthesis as well as recombinant methods.
U.S. Pat. No. 6,506,559 teaches methods for inhibiting gene expression in vitro using siRNA constructs that mediate RNAi. International Patent Publication Nos. WO 02/055692, WO02/055693, and WO 00/44895 describe certain methods for inhibiting gene expression using RNAi. International Patent Publication Nos. WO 99/49029 and WO 01/70949 describe various vectors expressing siRNA molecules. International Patent Publication No. WO 2006/060454 teaches methods of designing small interfering RNAs, antisense polynucleotides, and other hybridizing nucleotides. US Patent Application Publication No. 20060217331 discloses chemically modified double stranded nucleic acid molecules for RNA interference.
There remains an unmet need for therapeutic agents effective in attenuating or inhibiting cellular proliferation in particular for the treatment of hyper-proliferative disease. The art neither teaches nor suggests inhibiting cell proliferation by silencing VDAC1.